The long-term goal of this component of the Program Project is to elucidate the molecular basis of the pathogenic process in transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative diseases that afflict humans and animals. The protein-only model postulates that the infectious pathogen responsible for these diseases is a misfolded form of the prion protein, PrPSc, which self-propagates by binding to normal prion protein and catalyzing its conversion to the pathogenic form. TSE diseases have emerged as a major public health issue following recent epidemics of bovine spongiform encephalopathy (BSE) and indications that BSE might have crossed the species barrier to cause variant Creutzfeldt-Jakobdisease in humans. The main focus of this project is on understanding structural properties of various abnormally folded forms of the recombinaht prion protein. The first specific aim is to determine the structure of amyloid fibrils formed by the recombinant prion protein in vitro at a resolution close to amino acid residue. This will be accomplished using a number of newly emerged biophysical techniques that provide site- specific information about local protein mobility and accessibility and measurements of intermolecular and intramolecular distances between specific sites within the prion protein amyloid. Another specific aim is to expand and optimize the recently developed technique of protein misfolding by cyclic amplification (PMCA) to accomplish efficient brain PrP^-templated conversion of bacterially-expressed recombinant prion protein to proteinase K-resistantform, and assess the infectivity of this product in experimental animals. Finally, building on the experience gained in structural studies with spontaneously formed prion protein amyloid (Specific Aim 1), we will determine structural organization of the recombinant PrP oligomers generated in brain PrPSc- templated PMCA reaction. Our hypothesis is that the basic folding motif of PrPSc is similar to that of PrP amyloid fibrils, though some differences which may account for especially high resistance of brain PrPSc to proteolytic digestion are expected.